Antimalarial and antileishmanial activities of histone deacetylase inhibitors with triazole-linked cap group

Article abstract of DOI:10.1016/j.bmc.2009.10.042

Histone deacetylase inhibitors (HDACi) are endowed with plethora of biological functions including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. Parsing the structure-activity relationship (SAR) for each disease condition is vital for long-term therapeutic applications of HDACi. We report in the present study specific cap group substitution patterns and spacer-group chain lengths that enhance the antimalarial and antileishmanial activity of aryltriazolylhydroxamates-based HDACi. We identified many compounds that are several folds selectively cytotoxic to the plasmodium parasites compared to standard HDACi. Also, a few of these compounds have antileishmanial activity that rivals that of miltefosine, the only currently available oral agent against visceral leishmaniasis. The anti-parasite properties of several of these compounds tracked well with their anti-HDAC activities. The results presented here provide further evidence on the suitability of HDAC inhibition as a viable therapeutic option to curb infections caused by apicomplexan protozoans and trypanosomatids.

Full text of DOI:10.1016/j.bmc.2009.10.042

Bioorganic & Medicinal Chemistry 18 (2010) 415–425
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial and antileishmanial activities of histone deacetylase
inhibitors with triazole-linked cap group
Vishal Patil a,à, William Guerrant a,à, Po C. Chen a, , Berkley Gryder a, Derek B. Benicewicz a,
a,b,
Shabana I. Khan c, Babu L. Tekwani c, , Adegboyega K. Oyelere
*
*
a School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
b Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
c National Center for Natural Products Research and Department of Pharmacology, School of Pharmacy University of Mississippi, MS 38677-1848, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase inhibitors (HDACi) are endowed with plethora of biological functions including anti-
proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. Parsing the structure–
activity relationship (SAR) for each disease condition is vital for long-term therapeutic applications of
HDACi. We report in the present study specific cap group substitution patterns and spacer-group chain
lengths that enhance the antimalarial and antileishmanial activity of aryltriazolylhydroxamates-based
HDACi. We identified many compounds that are several folds selectively cytotoxic to the plasmodium
parasites compared to standard HDACi. Also, a few of these compounds have antileishmanial activity that
rivals that of miltefosine, the only currently available oral agent against visceral leishmaniasis. The anti-
parasite properties of several of these compounds tracked well with their anti-HDAC activities. The
results presented here provide further evidence on the suitability of HDAC inhibition as a viable thera-
peutic option to curb infections caused by apicomplexan protozoans and trypanosomatids.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 July 2009
Revised 19 October 2009
Accepted 23 October 2009
Available online 27 October 2009
Keywords:
Histone deacetylase
Malaria
Leishmaniasis
Plasmodium falciparum
Leishmania donovani
Aryltriazolylhydroxamates
Structure–activity relationship
1. Introduction
annually.6 Similarly, leishmaniasis is a tropical/sub-tropical para-
sitic disease, caused by the members of the genus Leishmania,
Inhibition of histone deacetylase (HDAC) activity has been lar-
gely validated as a viable therapeutic strategy for cancer treat-
ment.1 Of the several mechanisms proposed to explain the
molecular basis of the anti-proliferative activity of HDAC inhibitors
(HDACi), perturbation of chromatin remodeling and acetylation
states of key non-histone proteins have been widely accepted.2
Consequently, intense research activities are ongoing on the appli-
cation of HDACi to other diseases where chromatin remodeling and
protein acetylation states may play significant roles.3
Human pathogenic apicomplexan protozoans and trypanoso-
matids, such as the causative agents of malaria and leishmaniasis
respectively, have been shown to be responsive to HDACi.4 Malaria
is a serious infectious disease that is prevalent in sub- Saharan Afri-
ca and Asia.5 It is caused by members of the genus Plasmodium,
with an estimated 500 million infections and two million fatalities
which infects over two million people annually.7 These two dis-
eases constitute an emerging serious threat to public health due
to the emergence of multi-drug resistant strains and the occur-
rence of leishmania as opportunistic infective agents in human
immunodeficiency virus-infected patients.8–10 Therefore, there is
an urgent need for affordable alternative agents to curtail these
diseases.
Plasmodium falciparum, the principal malarial protozoan para-
site in humans, has at least five putative HDAC enzymes.11 Genes
encoding two of these putative HDAC—PfHDAC-1 and PfSir2—have
been partially characterized.11–13 Because of its homology with the
class I family of HDACs from human, chicken, frog and Saccharomy-
ces cerevisiae, PfHDAC-1 may be one of the intracellular targets
whose interaction with HDACi elicits the observed antimalarial
activity of HDACi.4c,f,g,11 Similarly, Leishmania parasite’s genome
contains multiple genes encoding different HDACs isozymes some
of which have been shown to be essential for the survival and pro-
liferation of Leishmania parasites.14–16 However, the structure–
activity relationship (SAR) of the antimalarial and antileishmanial
activities of HDACi is not entirely clear.4g
* Corresponding authors. Tel.: +1 662 915 7882; fax: +1 662 915 7062 (B.L.T.);
tel.: +1 404 894 4047; fax: +1 404 894 2291 (A.K.O.).
E-mail addresses: btekwani@olemiss.edu (B.L. Tekwani), aoyelere@gatech.edu
(A.K. Oyelere).
Previously, we and others have reported the synthesis and SAR
for aryltriazolylhydroxamates, HDACi incorporating 1,2,3-triazole
Present address: School of Medicinal Chemistry and Pharmacognosy, University
of Illinois at Chicago, USA.
4d,17
à
into the cap group-linking and surface recognition moieties.
These authors contributed equally to this manuscript.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.042

416
V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
We showed that these compounds displayed cap group- and
spacer-length dependent anti-HDAC and whole cell anti-prolifera-
tive activities that tracked with the three-motif pharmacophoric
model of all HDACi (Fig. 1).17 In this contribution, we sought to
clarify the specific structural attributes that confer antimalarial
and antileishmanial activities to aryltriazolylhydroxamates HDACi.
We found that for a given cap group, HDACi spacer-group length is
a major structural determinant of antimalarial and antileishmanial
activities. Specifically, the antimalarial and antileishmanial activi-
ties of these aryltriazolylhydroxamates generally peak in analogs
having 5 or 6 methylene spacer groups separating the active site
zinc binding hydroxamate moiety and the aryltriazolyl group.
amine under standard activation condition gave O-tritylated
hydroxamates 2a–c in good to excellent yields. Subsequent Cu(I)
catalyzed cycloaddition reaction between 2a–c and terminal al-
kynes 3a–g resulted in O-trityl protected aryltriazolylhydroxa-
mates 4–14.17 The unmasking of the O-trityl protection group
was facilitated by BF3ꢀOEt2 or TFA treatment to furnish the requi-
site compounds, 19, 20, 23, 24, 26, 27, 34, 42, 43, 47 and 50, all
new aryltriazolylhydroxamates having longer methylene spacer-
groups (n = 6–9) and varied HDAC surface recognition cap groups
(Scheme 1).
3. Results and discussion
3.1. In vitro assays
2. Chemistry
Our previous protocol for the synthesis of aryltriazolylhydroxa-
mates required the unmasking of the hydroxamic acid moiety of
the desired product from a penultimate ester intermediate by
treatment with aqueous hydroxylamine in the presence of a cata-
lytic amount of KCN.17 This reaction sometime gives the carboxylic
acid derivative of the desired product as a contaminant, thereby
complicating compound isolation. We describe here an alternative
route that allows for a facile synthesis of the aryltriazolylhydroxa-
mates of interest while avoiding the formation of carboxylic acid
contaminants. Reaction of azido acid 1a–c with O-tritylhydroxyl-
Our goal in the present study is to elucidate the structural
determinants that confer antimalarial and antileishmanial activi-
ties to aryltriazolylhydroxamates HDACi. To complement our pre-
vious studies on the anti-HDAC activities of this class of
compounds and also establish a SAR of their anti-protozoan activ-
ities, we synthesized additional aryltriazolylhydroxamates having
longer methylene spacer-groups (n = 6–9) and varied HDAC surface
recognition cap groups. We first evaluated the anti-HDAC activity
of the new compounds using the Fluor de Lys assay18 as described
previously.17,19a
Linker group
a
Zinc binding
group (ZBG)
Surface recognition
group
b
O
O
O
O
O
O
H
OH
N
OH
N
H
S
OH
N
N
H
N
H
H
O
N
TrichostatinA
(TSA)
suberoylanilide hydroxamic
acid (SAHA)
PXD-101
O
OH
O
OH
O
N
H
NH2
N
H
H
N
N
N
O
MS-275
N
LAQ-824
H
O
H
N
O
HN
OH
O
O
O
N
H
N
NH
NH
R
NH HN
O
O
O
O
O
H
O
N
HN
O
NH
O
O
S
O
Apicidin A (R = H)
Apicidin (R = OMe)
S
O
S
HO
N
FK-228
Tubacin
Figure 1. (a) A three-motif pharmacophoric model of HDAC inhibitors. (b) Selected examples of small molecule HDAC inhibitors.

V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
417
N
N
N
O
n
O
n
O
a
b
R
OTrityl
N3
N3
OTrityl
N
OH
N
H
n
H
1a: n = 6
2a: n = 6
4: n = 7, R = Ph
5: n = 8, R = Ph
10: n = 6, R = 2-Tol
11: n = 7, R = 3-Bp
12: n = 8, R = 3-Bp
1b
: n = 7
: n = 8
2b
: n = 7
: n = 8
1c
2c
6: n = 7, R = DMA
7
8
9
13
14
: n = 8, R = DMA
: n = 6, R = 3-Py
: n = 7, R = 3-Py
: n = 7, R = PyP
: n = 7, R = Nap
N
N
N
O
c
R
OH
N
n
H
19: n = 7, R = Ph
20: n = 8, R = Ph
34: n = 6, R = 2-Tol
42: n = 7, R = 3-Bp
23: n = 7, R = DMA 43: n = 8, R = 3-Bp
24
26
27
47
50
: n = 8, R = DMA
: n = 6, R = 3-Py
: n = 7, R = 3-Py
: n = 7, R = PyP
: n = 7, R = Nap
Scheme 1. Synthesis of aryltriazolylhydroxamates for SAR studies. Reagents and conditions: (a) NH2–O–trityl, IBCF, THF, ꢁ15 °C, 2 h; (b) 3a–g, CuI, Hunig’s base, THF, rt; (c)
BF3ꢀOEt2, THF, rt, 20 min or CH2Cl2, TFA/thioanisole (1:1), 0 °C, 3 h. Abbreviations: 3-Bp, 3-biphenyl; DMA, p-N,N-dimethylanilyl; Nap, 6-methoxynapthyl; Ph, phenyl; 3-Py, 3-
pyridyl; PyP, 4-pyridylphenyl; 2-Tol, 2-tolyl.
All aryltriazolylhydroxamates were tested for their ability to in-
duce in vitro inhibition of the proliferation of chloroquine-sensitive
(D6, Sierra Leone) and chloroquine-resistant (W2, Indochina)
strains of P. falciparum. The in vitro antileishmanial activities of
compounds were tested against the promastigote stage of Leish-
mania donovani, the causative agent of visceral leishmaniasis. Plas-
modium growth inhibition was determined by a parasite lactate
dehydrogenase assay using Malstat reagent,20 while inhibition of
viability of the promastigote stage of L. donovani was determined
using standard Alamar blue assay, modified to a fluorometric as-
say.21 Amphotericin B and pentamidine, standard antileishmanial
agents; chloroquine and artemisinin, standard antimalarials; and
suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA),
standard HDACi were all used as positive controls. To determine
selective toxicity index, all compounds were simultaneously tested
against a non-transformed mammalian cell line namely, monkey
kidney epithelial (Vero) using Neutral Red assay.22
Additionally, a similar spacer-length restriction in antimalarial
activity has been recently observed for acyl arylhydrazone based
compounds.4f Introduction of a N,N-dimethylamino moiety to the
para position of the cap group, similar to the substitution pattern
on TSA, had no effect on the methylene spacer length dependence
of the antimalarial activities. However, these N,N-dimethylamino
compounds have a relatively relaxed preference for either 5 or 6
methylene spacers (Table 1, comparing compounds 21 and 22).
Incorporation of nitrogen into the phenyl ring did not improve
the potency of the simple phenyl-substituted compounds. Never-
theless, the resulting pyridine derivatives 25–30 possessed anti-
malarial activity profile that was dependent on the ring location
of the nitrogen atom. For compounds with 5 methylene spacers,
the antimalarial activity was intolerant of nitrogen substitution
at the ortho position. This observation is contrary to the trend of
the anti-HDAC activities of these pyridyl compounds. However,
the ortho N-substitution intolerance was relieved with one addi-
tional methylene group (Table 1, comparing compounds 29 and
30). For compounds with the same pyridyl group, we observed
spacer length-dependent antimalarial activities in similar manner
to those of the unsubstituted phenyl analogs.
Other aryl group substitutions resulted in distinct antimalarial
activities that may give further indications of the electronic and
steric environment of the intracellular drug target(s) within the
parasite. Methyl substituted compounds 31–33 (n = 5) showed a
ring meta-position preference despite anti-HDAC activity that fa-
vored a ring ortho-position substitution. Extension of the linker
length by one methylene group drastically relieved the ortho-sub-
stitution intolerance of these series of methyl substituted com-
pounds (Table 1, comparing compounds 33 and 34). Conversely,
meta-substitution with a stronger electron-donating methoxy moi-
ety (relative to the methyl group) eliminated anti-parasite activity
while there was no clear preference between ortho- and para-sub-
stitution (Table 1, comparing compounds 35–37). This lack of pref-
erence is surprising considering the fact that these compounds had
anti-HDAC activity that was about 40-fold in favor of the para-
3.2. Structure–activity relationship
The HDAC inhibition profiles of the newly synthesized aryl-
triazolylhydroxamates showed a trend that paralleled those of
the previously reported compounds.17 In general; anti-HDAC activ-
ities are dependent on two key features of these molecules,
namely—the length of the methylene spacer-group (n) and the
identity of the cap group. Again, for a given cap group, the opti-
mum n is either 5 or 6 (Table 1).
The anti-protozoan activities of several of these aryl-
triazolylhydroxamates are also dependent on the two structural
features described above. For example, homologous compounds
15–20, derived from unsubstituted phenyl ring, have spacer
length-dependent antimalarial activities that peaked when n = 6
(i.e., 6 methylene spacers separating the triazole ring from the zinc
binding hydroxamic acid group) (Table 1). This antimalarial trend
is in close agreement with the anti-HDAC activities of these com-
pounds against HDACs 1 and 2 from HeLa cell nuclear fraction.17

418
V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
Table 1
In vitro HDAC Inhibition (nM), antilieshmanial (
l
g/mL) and antimalarial (ng/mL) activities of aryltriazolylhydroxamates HDACi
N
N
O
R
N
OH
N
n
H
Compd
R
n
HDAC inhibition Antileshmanial activityc
IC50 (nM)a
Antimalarial activityc
Cytotoxicity S. I. D6 (W2)
(VERO) IC50
(ng/mL)
IC50
(l
g/
IC90
(lg/
Plasmodium falciparum Plasmodium falciparum
(D6 clone) IC50 (ng/mL) (W2 clone) IC50 (ng/mL)
mL) SD
mL) SD
15
16
17
18
19
20
21
22
23
24
3
4
5
6
7
8
5
6
7
8
N.D.b
110.0b
14.2b
9.6b
NA
NA
2850 50
3500 800
120 10
130 30
24.5 1.5
695 115
2750 50
38.5 1.5
32 13
NC
NC
>1.7 (>1.4)
NA
NA
125
88
5
>38.08 (>39.6)
17.5 0.71
4.55 0.35
>40
2
5
3950 550 44.9 (30.4)
40.0 0.0
38
430 20
11.3 (17.6)
>7.4 (>6.8)
363.6
55.4
17.75 0.35 >40
645 55
NC
18.5 0.71
15.5 0.71
5.35 0.35
24.0 2.83
>40
>40
2650 450
3350 550 1.9 (1.2)
4.3b
68
8
3400 200 50.0 (88.3)
N
106.1b
15.9
36.0 1.41 24.5 5.5
765 35
970 130
NC
31.2 (23.9)
3.8 (3.6)
N
N
N
>40
175 105
185 15
1650 50
596.6
20.75 0.35 >40
11.45 3.61 >40
1800 500
>2.6 (>2.9)
25
26
5
6
287.2b
365.8
175 45
205 55
81 49
NC
>27.2 (>23.2)
N
N
N
15.5 0.71
40
75.5 21.5
3780 980 50.1 (46.7)
27
28
29
7
5
5
425.8
112.5b
67.6b
19.75 0.35 >40
17.75 0.35 >40
1495 253
195 65
985 15
1687 321
132.5 57.5
625 95
NC
NC
>3.2 (>2.8)
>24.4 (>35.9)
N
20.5 0.71
>40
2900 300 2.9 (4.6)
N

V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
419
Table 1 (continued)
Compd
R
n
HDAC inhibition Antileshmanial activityc
IC50 (nM)a
Antimalarial activityc
Cytotoxicity S. I. D6 (W2)
(VERO) IC50
(ng/mL)
IC50
(l
g/
IC90
(l
g/
Plasmodium falciparum Plasmodium falciparum
(D6 clone) IC50 (ng/mL) (W2 clone) IC50 (ng/mL)
mL) SD
mL) SD
30
31
6
5
23.9b
5.75 0.35
40.0 0.0
69
1
40
5
950 150
13.8 (23.8)
N
43.4b
31.9b
21.5 2.12
17.0 1.41
>40
>40
130 40
140 40
3850 650 29.6 (27.5)
2700 300 34.0 (39.7)
32
33
5
5
79.5 0.5
68
8
17.4b
23.0 1.41
>40
1250 50
120 20
1950 750
3300 700 2.6 (1.7)
34
35
6
5
2.8
26.0 0.0
40
140 20
90 10
710 210
5.9 (5.1)
2.1b
18.5 0.71
>40
130
NA
6
2350 850 18.1 (26.1)
O
36
37
5
5
13.9b
76.0b
NA
NA
NA
NC
—
O
18.75 0.35 >40
150 10
97.5 2.5
2300 500 15.3 (23.6)
O
O
O
38
5
315.9b
40
>40
>40
2000 300
2600 200
36.5 13.5
NC
>2.4 (>1.8)
39
40
5
5
31.7b
11.5 0.71
46
6
NC
NC
>103 (>130)
S
1.9b
11.25 0.35 32.5 0.71 37.5 17.5
26
25
4
>127 (>183.1)
41
42
6
7
5.4b
20.5 0.71
>40
25
5
5
NC
NC
>190.4 (>190.4)
108.9
20.25 0.35 40
840 40
650 50
>5.6 (>7.3)
(continued on next page)

420
V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
Table 1 (continued)
Compd
R
n
8
5
HDAC inhibition Antileshmanial activityc
IC50 (nM)a
Antimalarial activityc
Cytotoxicity S. I. D6 (W2)
(VERO) IC50
(ng/mL)
IC50
(l
g/
IC90
(lg/
Plasmodium falciparum Plasmodium falciparum
(D6 clone) IC50 (ng/mL) (W2 clone) IC50 (ng/mL)
mL) SD
mL) SD
43
147.7
20.0 0.0
40.0 0.0
3350 150
2100 800
3200 200
2600 100
NC
>1.4 (>1.5)
44
162.6b
13.0 2.83
38.0 0.0
2700 200 1.3 (1.0)
45
46
47
5
6
7
2.3b
28.0 2.83
>40
NA
NA
45.5 9.5
1150 150
88.5 6.5
47.5 22.5
1250 50
127.5 2.5
NC
NC
>104.6 (>100.2)
N
N
N
16.6b
212.9
NA
NA
>4.1 (>3.8)
3850 650 43.5 (30.2)
O
O
O
48
49
50
5
6
7
1.8b
18.0 2.83
18.5 2.12
NA
>40
>40
NA
35
5
31 11
2850 50
>81.4 (>91.9)
15.3b
226.1
47.5 4.5
53 13
2650 350 55.8 (50.0)
112 13
165 35
650 250
5.8 (3.9)
51
52
5
5
2.1b
9.5 0.71
39.0 1.41 33
3
34 16
2400 400 72.7 (70.6)
N
151.5b
18.5 0.71
>40
52.5 12.5
33
3
NC
>90.7 (>144.2)
N
Chloroquine
Artemisinin
Pentamidine
Amphotericine B
SAHA
—
—
—
—
—
—
NT
NT
NT
NT
65
5
NT
NT
0.90 0.9
0.14 0.01
29.7 5.4
1.37 0.16
NT
NT
17
4
NT
125
6
NT
NT
478.9 33.4
49.5 7.4
NT
NT
NT
NT
1200
95
NT
NT
NT
NT
4.8 (2.5)
2.6 (2.2)
1.80 0.0
0.31 0.01 NT
57.9 6.8
20.5 0.71 41.2 6.1
261.6 39.5
TSA
NA = not active up to the highest concentration tested.
NC = no cytotoxic up to highest concentration tested.
NT = not tested.
a
Each value is obtained from three independent experiments.
Cited from Ref. 17.
Values are mean SD of triplicates.
b
c
methoxy substitution (Table 1, comparing compounds 35 with 37).
However, bisortho methoxy-substitution, incorporated into com-
pound 38, led to a reduction of the antimalarial activity, an obser-
vation that may suggest that the parasite’s intracellular target(s)
may have similar steric constraints as the mammalian HDAC1.
Larger cap groups such as biphenyls, naphthalenes and quino-
lines also furnished compounds with potent antimalarial activities.
We observed that this class of compounds displayed spacer length-
dependent antimalarial activities in similar manner to their simple
aryl counterparts. Moreover, compounds’ antimalarial potency was

V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
421
dependent on the substitution pattern on these larger cap group
moieties. Similar to their anti-HDAC activities, the biphenyl com-
pounds 40–47 displayed varying antimalarial activities that were
dependent on the relative position of the triazole ring.17 Specifi-
cally, the meta-placement of the triazole ring was about 65–100-
fold preferred over an ortho-substitution by either strains of P. fal-
ciparum (Table 1, comparing compounds 40 and 44). While there
was no strong preference for either 5 or 6 methylene spacer group
among the meta-biphenyl compounds 40 and 41, 5 methylene-
linked 4(4-pyridyl) compound 45 was surprisingly 25-times more
potent compared to the 6 methylene congener 46. The antimalarial
activities of the 5 and 6 methylene spacer, six–six fused ring naph-
thalenes 48–49 and quinolines 51–52 were virtually indistinguish-
able. These compounds showed potent antimalarial activities with
IC50 values that were comparable to those of TSA and 5–15-fold
lower than those of SAHA against either strain of P. falciparum
(Table 1).
We also investigated the effects of these aryltriazolylhydroxa-
mates on the viability of the promastigote stage of L. donovani
and found that they have modest to moderate in vitro antileishma-
nial activities. Similar to their antimalarial effects, the antileishma-
nial activities of these compounds were generally maximal in
analogs with 5 or 6 methylene spacer groups. Worthy of specific
note are compounds 18, 22, 30, 39, 40 and 51 which inhibited
the proliferation of the promastigote stage of L. donovani with
IC50 values that were 2–4-fold lower than that of SAHA and compa-
rable to that of miltefosine, the only clinically approved oral drug
against visceral leishmaniasis.23,24 It is imperative to point out that
the antileishmanial activities of some of these compounds may be
primarily due to their general cytotoxicity at the measured IC50
values, in similar manner to that of SAHA.
and Analtech preparative TLC plates (UV 254, 2000
l
m) were used
for purification. UV light was used to examine the spots. Silica gel
(200–400 Mesh) was used in column chromatography. NMR
spectra were recorded on a Varian-Gemini 400 magnetic resonance
spectrometer. 1H NMR spectra were recorded in parts per
million (ppm) relative to the peak of CDCl3, (7.24 ppm), CD3OD
(3.31 ppm), or DMSO-d6 (2.49 ppm). 13C spectra were recorded rel-
ative to the central peak of the CDCl3 triplet (77.0 ppm), CD3OD
(49.0 ppm), or the DMSO-d6 septet (39.7 ppm), and were recorded
with complete heterodecoupling. Multiplicities are described using
the abbreviation s, singlet; d, doublet, t, triplet; q, quartet; m, mul-
tiplet; and app, apparent. High-resolution mass spectra were re-
corded at the Georgia Institute of Technology mass spectrometry
facility in Atlanta. Melting points (uncorrected) were recorded on
a Mel-Temp II apparatus. 7-Azidoheptanoic acid 1a was synthe-
sized starting from 7-bromoheptane nitrile while 8-azidooctanoic
acid 1b and 9-azidononanoic acid 1c were synthesized from the
corresponding x
-bromo acids as described previously.19a–c
4.2. Analogue synthesis
4.2.1. Representative procedure for conversion of
anoic acid to O-trityl protected hydroxamates. 7-Azido-O-trityl-
heptahydroxamate (2a)
x-azidoalk-
7-Azidoheptanoic acid 1a (434 mg, 2.54 mmol) was dissolved in
anhydrous THF. N-Methylmorpholine (257 mg, 2.54 mmol) was
added to the solution. The reaction mixture was then cooled down
to ꢁ15 °C and stirred for 5 min. Isobutylchloroformate (346 mg,
2.55 mmol) was added and the mixture was stirred for 10 min at
ꢁ15 °C. O-Tritylhydroxylamine (700 mg, 2.55 mmol) was added
followed by 2 more equivalents of N-methylmorpholine. Stirring
continued for 15 min at ꢁ15 °C and 2 h at room temperature.
Afterwards the mixture was poured into 2 M HCl and extracted
three times in each case with water, sodium bicarbonate solution
(5%) and water. After washing with brine and drying over Na2SO4,
solvent was evaporated in vacuo. Column chromatography using
25% EtOAc in hexanes as the eluent system gave compound 2
837 mg (77%) as a white solid. 1H NMR (CDCl3, 400 MHz) d 0.91–
0.99 (2H, m), 1.11–1.21 (4H, m), 1.44 (2H, p, J = 7.6 Hz), 1.77 (2H,
t, J = 7.2 Hz), 3.24 (2H, t, J = 6.8 Hz), 7.29–7.38 (15H, m); 13C NMR
(CDCl3, 100 MHz) d 23.0, 26.1, 28.3, 30.8, 51.0, 93.0, 127.3, 127.9,
128.8, 140.9, 176.9.
Finally, several of the compounds that potently inhibited para-
site proliferation were found to be less cytotoxic to normal mam-
malian Vero cells. For example, 18 compounds—16, 17, 21, 22,
25, 26, 28, 31, 32, 39–41, 45, 47–49, 51 and 52—were several folds
selectively toxic to plasmodium parasite compared to the two
standard HDAC inhibitors—SAHA and TSA (Table 1).
3.3. Conclusion
We have identified in the present study specific cap group sub-
stitution patterns and spacer-group chain lengths that enhanced
the antimalarial and antileishmanial activities to aryltriazolylhydr-
oxamates HDACi. The anti-parasite properties of several of these
compounds tracked well with their anti-HDAC activities. Many of
the compounds herein disclosed are several folds selectively cyto-
toxic to the Plasmodium parasite compared to standard HDACi.
Also, a few of these compounds have antileishmanial activities that
rivaled the only currently available oral agent against visceral
leishmaniasis. Our results provide additional evidence on the suit-
ability of HDAC inhibition as a viable therapeutic approach to cur-
tail infections caused by apicomplexan protozoans and
trypanosomatids. The remarkable and selective anti-parasitic
properties of several of these aryltriazolylhydroxamates warrant
further investigation. A few lead compounds are being advanced
further for in vivo evaluation in murine malarial models.
4.2.2. 8-Azido-O-trityloctahydroxamate (2b)
Reaction of 8-azidooctanoic acid 1b (1.71 g, 9.21 mmol) and O-
tritylhydroxylamine (2.55 g, 9.27 mmol) as described for the syn-
thesis of 2a, followed by flash chromatography (eluent 2:1 hex-
anes/EtOAc) gave 2.59 g (88%) of 2b as a white solid. 1H NMR
(CDCl3, 400 MHz) d 0.88–1.39 (8H, m), 1.39–1.54 (4H, m), 3.12
(2H, t, J = 6.9 Hz), 7.10–7.49 (15H, m), 7.67 (1H, s); 13C NMR (CDCl3,
100 MHz) d 23.2, 24.9, 26.4, 28.7, 31.1, 33.2, 51.3, 93.1, 127.1,
128.0, 128.9, 141.0, 141.8, 146.8, 177.1.
4.2.3. 9-Azido-O-tritylnonahydroxamate (2c)
Reaction of 9-azidononanoic acid 1c (724 mg, 3.63 mmol) and
O-tritylhydroxylamine (1.00 g, 3.63 mmol) overnight as described
for the synthesis of 2a, followed by flash chromatography (eluent
2:1 hexanes/EtOAc) gave 940 mg (56%) of 2c as a sticky white solid.
1H NMR (CDCl3, 400 MHz) d 1.26 (10H, m), 1.57 (4H, m), 3.24 (2H, t,
J = 6.8 Hz), 7.34 (15H, m), 7.74 (1H, s).
4. Experimental
4.1. General
4.2.4. Representative procedure for Cu(I)-catalyzed cycloaddi-
tion reaction. O-Trityl-3-pyridyltriazolylheptahydroxamate (8)
7-Azido-O-tritylheptahydroxamate 2a (207 mg, 0.48 mmol) and
3-ethynylpyridine (50 mg, 0.48 mmol) were dissolved in anhy-
drous THF (10 mL) and stirred under argon at room temperature.
x
-Bromoalkanoic acids and 7-bromoheptane nitrile were pur-
chased from Sigma–Aldrich. Anhydrous solvents and other re-
agents were purchased and used without further purification.
Analtech silica gel plates (60 F254) were used for analytical TLC,

422
V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
Copper(I) iodide (9 mg, 0.05 mmol) and Hunig’s base (0.1 mL) were
then added to the reaction mixture, and stirring continued for 24 h.
The reaction mixture was diluted with CH2Cl2 (30 mL) and washed
with 1:4 NH4OH/saturated NH4Cl (3 ꢂ 30 mL) and saturated NH4Cl
(30 mL). The organic layer was dried over Na2SO4 and concentrated
in vacuo. The crude product was purified by flash chromatography
(gradient 3:1 CH2Cl2/acetone, then CH2Cl2/MeOH (5%)) to give
206 mg (81%) of 8 as a white solid. 1H NMR (DMSO-d6, 400 MHz)
d 0.94–1.01 (2H, m), 1.06–1.19 (4H, m), 1.71–1.78 (4H, m), 3.59
(1H, s), 4.34 (2H, t, J = 6.8 Hz), 7.25–7.36 (16H, m), 7.44–7.47 (1H,
m), 8.07 (1H, s), 8.17–8.20 (1H, m), 8.51–8.52 (1H, m), 8.68 (1H,
s), 9.03–9.04 (1H, m), 10.16 (1H, s); 13C NMR (CDCl3, 100 MHz) d
25.8, 29.7, 30.7, 46.9, 50.1, 53.8, 119.8, 123.5, 126.6, 127.7, 128.4,
128.8, 132.7, 144.0, 144.4, 146.7, 148.9, 176.8.
scribed for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 5:3 CH2Cl2/acetone) gave 170 mg (83.3%) of
9 as a white solid. 1H NMR (CD3OD, 400 MHz) d 0.90–1.06 (2H,
m), 1.09–1.38 (6H, m), 1.77–2.00 (4H, m), 4.38 (2H, t, J = 7.3 Hz),
7.10–7.59 (15H, m), 8.22 (1H, d, J = 8.3 Hz), 8.44 (1H, s), 8.46 (1H,
d, J = 4.8 Hz), 8.99 (1H, d, J = 2.3 Hz); 13C NMR (CD3OD, 100 MHz)
d 24.5, 26.2, 27.2, 29.6, 31.1, 33.4, 51.6, 94.2, 123.0, 125.6, 128.7,
130.4, 134.8, 143.5, 145.2, 147.2, 149.5, 173.0.
4.2.10. O-Trityl-2-tolyltriazolylheptahydroxamate (10)
Reaction of 7-azido-O-tritylheptahydroxamate 2a (147 mg,
0.34 mmol) and 2-ethynyltoluene (40 mg, 0.34 mmol) as described
for synthesis of 8, followed by flash chromatography (gradient
CH2Cl2/acetone 7:1, 6:1, 5:1) gave 160 mg (85%) of compound 10
as white solid.. 1H NMR (DMSO-d6, 400 MHz) d 0.94–1.01 (2H,
m), 1.07–1.19 (4H, m), 1.72–1.79 (4H, m), 2.40 (3H, s), 4.33 (2H,
t, J = 7.2 Hz), 7.22–7.30 (17H, m), 7.70–7.74 (1H, m), 8.35 (1H, s),
10.16 (1H, s); 13C NMR (DMSO-d6, 100 MHz) d 21.1, 25.5, 27.6,
29.5, 31.8, 49.3, 54.8, 91.6, 123.0, 125.9, 127.3, 127.4, 127.6,
128.1, 128.9, 130.1, 130.8, 134.8, 142.4, 145.4, 170.2.
4.2.5. O-Tritylphenyltriazolyloctahydroxamate (4)
Reaction of 8-azido-O-trityloctahydroxamate 2b (150 mg,
0.34 mmol) and phenylacetylene (35.9 mg, 0.35 mmol) as de-
scribed for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 1:1 hexanes/EtOAc) gave 90 mg (48.6%) of 4
as a colorless solid. 1H NMR (CDCl3, 400 MHz) d 0.94–1.12 (2H,
m), 1.12–1.46 (6H, m), 1.49–1.65 (2H, m), 1.75–1.97 (2H, m),
4.33 (2H, t, J = 7.3 Hz), 7.17–7.64 (18H, m), 7.72 (1H, s), 7.75 (1H,
s), 7.81 (2H, d, J = 7.5); 13C NMR (CDCl3, 100 MHz) d 23.2, 24.7,
26.1, 28.7, 31.0, 33.1, 50.3, 93.0, 119.3, 125.7, 128.0, 128.9, 130.6,
141.0, 141.7, 147.7, 178.2.
4.2.11. O-Trityl-3-biphenyltriazolyloctahydroxamate (11)
Reaction of 8-azido-O-trityloctahydroxamate 2b (137 mg,
0.31 mmol) and 1,10-biphenyl-3-ethynyl (60 mg, 0.34 mmol) as de-
scribed for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 18:1 CH2Cl2/acetone) gave 170 mg (88.5%) of
11 as a white solid. 1H NMR (CDCl3, 400 MHz) d 0.92–1.12 (2H,
m), 1.12–1.47 (6H, m), 1.75–1.97 (4H, m), 4.34 (2H, t, J = 7.1 Hz),
7.20–7.39 (13H, m), 7.40–7.52 (5H, m), 7.54 (2H, d, J = 7.7 Hz),
7.64 (2H, d, J = 7.4 Hz), 7.77 (1H, s), 7.78 (2H, m), 8.06 (1H, s);
13C NMR (CDCl3, 100 MHz) d 23.2, 24.9, 26.1, 28.5, 30.9, 33.0,
50.4, 93.0, 119.6, 124.5, 126.8, 127.1, 127.4, 128.1, 128.7, 129.0,
129.2, 131.1, 140.7, 141.0, 141.7, 147.6, 177.4.
4.2.6. O-Tritylphenyltriazolylnonahydroxamate (5)
Reaction of 9-azido-O-tritylnonahydroxamate 2c (80.4 mg,
0.18 mmol) and phenylacetylene (23.5 mg, 0.23 mmol) as de-
scribed for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 11:10 hexanes/EtOAc) gave 72.0 mg (73.2%)
of 5 as a sticky white solid. 1H NMR (CDCl3, 400 MHz) d 0.88–
1.44 (12H, m), 1.89 (2H, m), 4.34 (2H, t, J = 7.1 Hz), 7.15–7.6
(18H, m), 7.72 (1H, s), 7.79–7.81 (2H, d, J = 8 Hz); 13C NMR (CDCl3,
100 MHz) d 23.6, 26.6, 28.9, 29.2, 30.5, 31.2, 31.4, 50.6, 119.7,
125.9, 128.3, 129.0, 129.3, 141.3, 147.9, 177.5.
4.2.12. O-Trityl-3-biphenyltriazolylnonahydroxamate (12)
Reaction of 9-azido-O-tritylnonahydroxamate 2c (130 mg,
0.29 mmol) and 1,10-biphenyl-3-ethynyl (65 mg, 0.37 mmol) as de-
scribed for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 30:1 CH2Cl2/MeOH) gave 144.2 mg (81.5%)
of 11 as a red-brown solid. 1H NMR (CDCl3, 400 MHz) d 0.9–1.2
(6H, m), 1.21–1.42 (6H, m), 1.86 (2H, m), 4.31 (2H, m), 7.16–7.47
(19H, m), 7.51 (1H, d, J = 8.0 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.76 (3H,
m), 8.05 (1H, s); 13C NMR (CDCl3, 100 MHz) d 23.6, 26.6, 29.0,
29.2, 30.0, 30.5, 31.4, 50.6, 119.9, 124.7, 124.8, 127.1, 127.4,
128.1, 128.2, 128.4, 129.0, 129.3, 129.5, 131.5, 141.0, 142.0,
147.8, 177.6.
4.2.7. O-Trityl-p-N,N-dimethylanilyltriazolyloctahydroxamate (6)
Reaction of 8-azido-O-trityloctahydroxamate 2b (514 mg,
1.16 mmol) and 4-ethynyl-N,N-dimethylanaline (169 mg, 1.16
mmol) as described for the synthesis of 8, followed by purification
using preperative TLC (eluent 8:1 CH2Cl2/acetone) gave 560 mg
(82.1%) of 6 as a white solid. 1H NMR (CD3OD, 400 MHz) d 0.93–
1.06 (2H, m), 1.11–1.36 (6H, m), 1.76–1.92 (4H, m), 2.92 (6H, s),
4.31 (2H, t, J = 7.0 Hz), 6.76 (2H, d, J = 9.0 Hz), 7.18–7.43 (15H, m),
7.62 (2H, d, J = 9.0 Hz), 8.05 (1H, s); 13C NMR (CD3OD, 100 MHz) d
24.5, 26.2, 27.3, 29.6, 31.1, 33.4, 40.8, 51.3, 94.3, 113.8, 119.8,
120.5, 127.6, 128.6, 130.4, 143.6, 149.4, 152.1, 173.2.
4.2.13. O-Trityl-4-pyridylphenyltriazolyloctahydroxamate (13)
Reaction of 8-azido-O-trityloctahydroxamate 2b (150 mg,
0.34 mmol) and 4-(4-ethynylphenyl)-pyridine (61 mg, 0.34 mmol)
as described for synthesis of 8, followed by purification using pre-
parative TLC (eluent 10:1 Et2O/EtOH) gave 133 mg (63%) of com-
pound 13 as white solid. 1H NMR (DMSO-d6, 400 MHz) d 0.86–
0.92 (2H, m), 1.10–1.20 (6H, m), 1.73–1.81 (4H, m), 4.37 (2H, t,
J = 6.8 Hz), 7.26–7.34 (15H, m), 7.74 (2H, d, J = 6.0 Hz), 7.89 (2H,
d, J = 8.4 Hz), 7.97 (2H, d, J = 8.4 Hz), 8.62–8.64 (2H, m), 8.67 (1H,
s), 10.1 (1H, s); 13C NMR (CDCl3, 100 MHz) d 23.1, 26.1, 28.5,
28.7, 29.6, 30.1, 50.3, 119.7, 121.2, 123.6, 126.2, 127.3, 127.8,
128.5, 128.9, 131.4, 137.3, 140.9, 144.1, 146.7, 147.5, 150.1, 177.1.
4.2.8. O-Trityl-p-N,N-dimethylanilyltriazolylnonahydroxamate
(7)
Reaction of 9-azido-O-tritylnonahydroxamate 2c (100 mg,
0.22 mmol) and 4-ethynyl-N,N-dimethylanaline (49 mg, 0.34 mmol)
as described for the synthesis of 8, followed by purification using pre-
perative TLC (eluent 40:1:0.1 CH2Cl2/MeOH/NH4OH) gave 72.5 mg
(55%) of 7 as a white solid. 1H NMR (CDCl3, 400 MHz) d 1.04–1.60
(12H, m), 1.88 (2H, m), 4.32 (2H, t, J = 7.2 Hz), 6.74–6.76 (2H, d,
J = 8.8 Hz), 7.25–7.53 (16H, m), 7.58 (1H, s), 7.66–7.69 (2H, d,
J = 8.8 Hz); 13C NMR (CDCl3, 100 MHz) d 23.6, 26.6, 29.0, 29.2, 29.9,
30.5, 31.4, 40.7, 50.5, 112.7, 118.2, 119.2, 126.9, 127.0, 128.3, 129.3,
141.3, 142.1, 148.4, 149.5, 150.6, 177.5.
4.2.14. O-Trityl-6-methoxynapthyltriazolyloctahydroxamate (14)
Reaction of 8-azido-O-trityloctahydroxamate 2b (135 mg, 0.30
mmol) and 2-ethynyl-6-methoxynapthalene (56 mg, 0.30 mmol)
as described for synthesis of 8, followed by purification using
preparative TLC (eluent 25:1 Et2O/EtOH) gave 142 mg (76%) of
compound 14 as white solid. 1H NMR (DMSO-d6, 400 MHz)
4.2.9. O-Trityl-3-pyridyltriazolyloctahydroxamate (9)
Reaction of 8-azido-O-trityloctahydroxamate 2b (165 mg,
0.37 mmol) and 3-ethynylpyridine (38.3 mg, 0.37 mmol) as de-

V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
423
d 0.86–0.95 (2H, m), 1.10–1.21 (3H, m), 1.73–1.81 (2H, m), 3.86 (3H,
s), 4.37 (2H, t, J = 6.8 Hz), 7.15–7.32 (17H, m), 7.83–7.92 (3H, m), 8.29
(1H, m), 8.61–8.62 (1H, m), 10.12 (1H, s); 13C NMR (CDCl3, 100 MHz)
d 23.1, 26.1, 28.5, 28.7, 29.6, 30.1, 50.3, 55.2, 93.6, 105.7, 119.1, 119.3,
124.1, 124.3, 125.8, 127.2, 127.8, 127.8, 128.0, 128.9, 128.9, 129.6,
134.2, 140.9 147.8, 157.8, 177.1.
4.2.19. 7-(2-Tolyl)triazolylheptahydroxamamic acid (34)
Reaction of O-trityl-2-tolyltriazolylheptahydroxamate 10
(90 mg, 0.16 mmol) with TFA (0.2 mL) and thioanisole (0.2 mL) in
CH2Cl2 (7 mL) at 0 °C within 3 h as described for the synthesis of
26, followed by purification using preparative TLC (eluent
12:1:0.2 CH2Cl2/MeOH/NH4OH) gave 46 mg (93%) of 34 as an off-
white semi-solid. 1H NMR (CDCl3, 400 MHz) d 1.26–1.34 (4H, m),
1.53–1.61 (2H, m), 1.84–1.91 (2H, m), 2.05–2.14 (2H, m), 2.40
(3H, s), 4.34 (2H, t, J = 6.8 Hz), 7.20–7.22 (3H, m), 7.64 (1H, s),
7.67–7.70 (1H, m), 9.72 (1H, s); 13C NMR (DMSO-d6, 100 MHz) d
21.3, 24.9, 25.6, 27.8, 29.6, 29.8, 50.0, 121.9, 126.0, 128.1, 128.7,
129.7, 130.8, 135.4, 146.9, 171.3. HRMS (FAB, thioglycerol) calcd
for [C16H22N4O2+H]+ 303.1821, found 303.1810.
4.2.15. Representative procedure for conversion of O-tritylhydr-
oxamate to hydroxamic acid via TFA deprotection. 7-(3-Pyridyl)-
triazolylheptahydroxamic acid (26)
To a solution of O-trityl-3-pyridyltriazolylheptahydroxamate 8
(90 mg, 0.16 mmol) in CH2Cl2 (7 mL) was added trifluoroacetic acid
(TFA) (0.2 mL) and thioanisole (0.2 mL) dropwise at 0 °C. Reaction
mixture stirred for next 3 h at 0 °C. Solvent evaporated off in vacuo.
The crude mixture was loaded on preparative TLC, eluted with
12:1:0.2 CH2Cl2/MeOH/NH4OH to give 39 mg (84%) of 26 as a white
solid; mp 146–148 °C. 1H NMR (DMSO-d6, 400 MHz) d 1.21–1.29
(4H, m), 1.46 (2H, m), 1.84 (2H, m), 1.91 (2H, m), 4.39 (2H, t,
J = 7.2 Hz), 7.45–7.48 (1H, m), 8.18 (1H, m), 8.52 (1H, s), 8.65
(1H, s), 8.70 (1H, s), 9.03 (1H, s), 10.31 (1H, s). 13C NMR (DMSO-
d6, 100 MHz) d 24.9, 25.5, 27.9, 29.4, 32.1, 49.5, 121.9, 124.0,
132.4, 143.4, 146.2, 148.7, 169.0. HRMS (FAB, thioglycerol) calcd
for [C14H19N5O2+H]+ 290.1617, found 290.1615.
4.2.20. 8-(3-Biphenyl)triazolyloctahydroxamic acid (42)
Reaction of O-trityl-3-biphenyltriazolyloctahydroxamate 11
(170 mg, 0.23 mmol) with TFA (0.1 mL) and thioanisole (0.1 mL)
in CH2Cl2 (10 mL) at 0 °C within 3 h as described for the synthesis
of 26, followed by purification using preparative TLC (eluent 12:1
CH2Cl2/MeOH) gave 80 mg (77.2%) of 42 as a white solid; mp
110–112 °C. 1H NMR (CD3OD, 400 MHz) d 1.14–1.44 (6H, m),
1.45–1.66 (2H, m), 1.78–1.96 (2H, m), 1.96–2.13 (2H, m), 4.35
(2H, t, J = 6.9 Hz), 7.31 (1H, t, J = 6.7 Hz), 7.41 (2H, t, J = 7.6 Hz),
7.46 (1H, d, J = 7.3 Hz), 7.55 (1H, d, J = 7.3 Hz), 7.63 (2H, d,
J = 7.3 Hz), 7.75 (1H, d, J = 7.3 Hz), 8.07 (1H, s), 8.33 (1H, s) 13C
NMR (CD3OD, 100 MHz) d 26.5, 27.3, 29.6, 29.8, 31.1, 33.7, 51.4,
122.4, 125.1, 125.5, 127.8, 128.0, 128.6, 129.9, 130.5, 132.2,
141.8, 143.1, 148.6, 172.9. HRMS (FAB, thioglycerol) calcd for
[C22H26N4O2+H]+ 379.2134, found 379.2139.
4.2.16. 8-(Phenyl)triazolyloctahydroxamic acid (19)
Reaction of O-trityl-phenyltriazolyloctahydroxamate 4 (70 mg,
0.13 mmol) with TFA (0.1 mL) and thioanisole (0.1 mL) in CH2Cl2
(10 mL) at 0 °C within 3 h as described for the synthesis of 26, fol-
lowed by purification using preparative TLC (eluent 8:1 CH2Cl2/
MeOH) gave 20 mg (51%) of 19 as a white solid; mp 118–121 °C.
1H NMR (CD3OD, 400 MHz) d 1.22–1.48 (6H, m), 1.52–1.70 (2H,
m), 1.87–2.01 (2H, m), 2.02–2.18 (2H, m), 4.44 (2H, t, J = 7.1 Hz),
7.34 (1H, t, J = 7.2 Hz), 7.43 (2H, t, J = 7.9 Hz), 7.81 (2H, d,
J = 7.2 Hz), 8.32 (1H, s); 13C NMR (DMSO-d6, 100 MHz) d 26.5,
27.2, 29.6, 29.8, 31.1, 33.6, 51.4, 122.2, 126.6, 129.3, 130.0, 131.7,
148.8, 169.1. HRMS (FAB, thioglycerol) calcd for [C16H22N4O2+H]+
303.1821, found 303.1806.
4.2.21. 9-(3-Biphenyl)triazolylnonahydroxamic acid (43)
Reaction of O-trityl-3-biphenyltriazolylnonahydroxamate 12
(114 mg, 0.18 mmol) with TFA (0.25 mL) and thioanisole
(0.25 mL) in CH2Cl2 (10 mL) at 0 °C within 3 h as described for
the synthesis of 26, followed by purification using preparative
TLC (eluent 20:1 CH2Cl2/MeOH) gave 63.4 mg (91%) of 43 as a
white solid; mp 122–125 °C. 1H NMR (DMSO-d6, 400 MHz) d 1.23
(8H, m), 1.42 (2H, m), 1.88 (4H, m), 4.36 (2H, m), 7.36 (1H, t,
J = 7.6 Hz), 7.42–7.54 (3H, m), 7.59 (1H, d, J = 7.2 Hz), 7.69 (2H, d,
J = 7.6 Hz), 7.82 (1H, d, J = 7.6 Hz), 8.08 (1H, s), 8.62 (1H, s), 8.68
(1H, s), 10.29 (1H, s); 13C NMR (DMSO-d6, 100 MHz) d 25.8, 26.5,
29.0, 29.1, 29.2, 30.3, 50.2, 122.3, 124.0, 124.7, 126.8, 127.4,
128.3, 129.6, 130.2, 132.1, 140.5, 141.4, 146.9, 167.9. HRMS (FAB,
thioglycerol) calcd for [C23H28N4O2+H]+ 393.2246, found 393.2238.
4.2.17. 9-(Phenyl)triazolylnonahydroxamic acid (20)
Reaction of O-trityl-phenyltriazolylnonahydroxamate 5 (57 mg,
0.10 mmol) with TFA (0.1 mL) and thioanisole (0.1 mL) in CH2Cl2
(10 mL) at 0 °C within 3 h as described for the synthesis of 26, fol-
lowed by purification using preparative TLC (eluent 18:1:0.1
CH2Cl2/MeOH/NH4OH) gave 9.9 mg (27.5%) of 20 as a white solid;
mp 135–137 °C. 1H NMR (DMSO-d6, 400 MHz) d 1.12–1.3 (8H, m),
1.42 (2H, m), 1.81 (2H, m), 1.87 (2H, t, J = 7.2 Hz), 4.34 (2H, t,
J = 7.0 Hz), 7.28 (1H, t, J = 7.4 Hz), 7.40 (2H, t, J = 7.4 Hz), 7.80 (2H,
d, J = 8.2 Hz), 8.54 (1H, s), 8.61 (1H, s), 10.28 (1H, s); 13C NMR
(DMSO-d6, 100 MHz) d 25.7, 26.5, 29.0, 29.15, 29.22, 30.3, 32.9,
50.2, 121.9, 125.8, 128.4, 129.6, 131.5, 146.9, 169.8. HRMS (FAB,
thioglycerol) calcd for [C17H24N4O2+H]+ 317.1933, found 317.1984.
4.2.22. 8-(4-Pyridylphenyl)triazolyloctahydroxamamic acid
(47)
Reaction of O-trityl-4-pyridylphenyltriazolyloctahydroxamate
13 (80 mg, 0.12 mmol) with TFA (0.2 mL) and thioanisole
(0.2 mL) in CH2Cl2 (7 mL) at 0 °C within 3 h as described for the
synthesis of 26, followed by purification using preparative TLC
(eluent 12:1:0.2 CH2Cl2/MeOH/NH4OH) gave 29 mg (61%) of 47
as a white solid; mp 152–154 °C. 1H NMR (DMSO-d6, 400 MHz) d
1.21–1.26 (6H, m), 1.45 (2H, p, J = 7.2 Hz), 1.83–1.93 (4H, m),
3.86 (3H, s), 4.39 (2H, t, J = 7.2 Hz), 7.75–7.81 (2H, m), 7.91–7.93
(2H, m), 7.98–8.00 (2H, m), 8.62–8.70 (3H, s), 10.34 (1H, s); 13C
NMR (DMSO-d6, 100 MHz) d 25.0, 25.7, 28.0, 28.3, 29.5, 32.2,
49.5, 121.1, 121.8, 125.7, 127.4, 131.8, 136.1, 145.6, 146.6, 150.0,
169.1. HRMS (FAB, thioglycerol) calcd for [C21H26N5O2+H]+
380.2086, found 380.2102.
4.2.18. 9-(p-N,N-Dimethylanilyl)triazolylnonahydroxamic acid
(24)
Reaction of O-trityl-(p-N,N-dimethylanilyl)triazolylnonahydr-
oxamate 7 (65.3 mg, 0.11 mmol) with TFA (0.1 mL) and thioanisole
(0.1 mL) in CH2Cl2 (10 mL) at 0 °C within 3 h as described for the
synthesis of 26, followed by purification using preparative TLC
(eluent 40:1 CH2Cl2/MeOH) gave 28.1 mg (72%) of 24 as a white so-
lid; mp 106–108 °C. 1H NMR (DMSO-d6, 400 MHz) d 1.21 (8H, m),
1.44 (2H, m), 1.81 (2H, m), 1.90 (2H, t, J = 7.2 Hz), 4.31 (2H, t,
J = 7.2 Hz), 6.75 (2H, d, J = 8.8 Hz), 7.62 (2H, d, J = 8.8 Hz), 8.33
(1H, s), 8.64 (1H, s), 10.31 (1H, s); 13C NMR (DMSO-d6, 100 MHz)
d 25.7, 26.6, 29.0, 29.16, 29.24, 30.3, 32.9, 40.7, 50.0, 113.0,
119.5, 120.0, 126.7, 147.5, 150.6, 169.8. HRMS (FAB, thioglycerol)
calcd for [C19H29N5O2+H]+ 360.2400, found 360.2402.
4.2.23. 8-(6-Methoxynapthyl)triazolylheptaoctahydroxamamic
acid (50)
Reaction of 6-methoxynapthyltriazolyloctahydroxamate 14
(90 mg, 0.14 mmol) with TFA (0.2 mL) and thioanisole (0.2 mL) in
CH2Cl2 (7 mL) at 0 °C within 3 h as described for the synthesis of

424
V. Patil et al. / Bioorg. Med. Chem. 18 (2010) 415–425
26, followed by purification using preparative TLC (eluent 12:1:0.2
CH2Cl2/MeOH/NH4OH) gave 34 mg (64%) of 50 as a white solid; mp
166–168 °C. 1H NMR (DMSO-d6, 400 MHz) d 1.20–1.26 (6H, m),
1.45 (2H, p, J = 7.2 Hz), 1.83–1.92 (4H, m), 3.86 (3H, s), 4.38 (2H,
t, J = 7.2 Hz), 7.16 (1H, dd, J = 2.3 Hz, 9.0 Hz), 7.31 (1H, d,
J = 2.3 Hz), 7.84–7.92 (2H, m), 8.29 (1H, s), 8.62 (1H, s), 8.64 (1H,
s), 10.31 (1H, s). 13C NMR (DMSO-d6, 100 MHz) d 24.9, 25.7, 28.0,
28.3, 29.6, 32.1, 49.4, 55.2, 105.9, 119.1, 121.1, 123.3, 124.1,
126.0, 127.3, 133.8, 146.4, 157.3, 169.0. HRMS (FAB, thioglycerol)
calcd for [C21H26N4O3+H]+ 383.2083, found 383.2104.
IndoChina) strains of P. falciparum. The 96 well microplate assay
is based on evaluation of the effect of the compounds on growth
of asynchronous cultures of P. falciparum, determined by the assay
of parasite lactate dehydrogenase (pLDH) activity.20 The appropri-
ate dilutions of the compounds were prepared in DMSO or RPMI-
1640 medium and added to the cultures of P. falciparum (2%
hematocrit, 2% parasitemia) set-up in clear flat bottomed 96 well
plates. The plates were placed into the humidified chamber and
flushed with a gas mixture of 90% N2, 5% CO2 & 5% O2. The cultures
were incubated at 37 °C for 72 h. Growth of the parasite in each
well was determined by pLDH assay using MalstatÒ reagent.20
The medium and RBC controls were also set-up in each plates.
The standard antimalarial agents, chloroquine and artemisinin,
were used as the positive controls while DMSO was tested as the
negative control. Antileishmanial activity of the compounds was
tested in vitro on a culture of L. donovani promastigotes. In a 96
well microplate assay compounds with appropriate dilution were
added to the leishmania promastigotes culture (2 ꢂ 106 cell/mL).
The plates were incubated at 26 °C for 72 h and growth of leish-
mania promastigotes was determined by Alamar blue assay.21
Pentamidine and Amphotericin B were tested as standard antile-
ishmanial agents. All the compounds were simultaneously tested
for cytotoxicity on VERO (monkey kidney epithelial) cells by Neu-
tral Red assay.22 IC50 value for each compound was computed from
the growth inhibition curve.
4.2.24. Representative procedure for conversion of
O-tritylhydroxamate to hydroxamic acid via BF3ꢀOEt2
deprotection. 8-(3-Pyridyl)triazolyloctahydroxamic acid (27)
To a solution of O-trityl-3-pyridyltriazolyloctahydroxamate 9
(121 mg, 0.22 mmol) in just enough THF to dissolve (ca. 1 mL)
was added thioanisole (0.31 mL) followed by BF3ꢀOEt2 (0.34 mL)
dropwise at rt. The reaction mixture was stirred for 20 min during
which a quantitative deprotection was observed bt TLC. The mix-
ture was poured into 20% MeOH in EtOAc (40 mL) and washed with
water (10 mL). The pH of the aqueous layer was adjusted to ꢃ7 and
extracted with 20% MeOH in EtOAc (3 ꢂ 50 mL). The combined or-
ganic layer was dried over Na2SO4 and concentrated in vacuo. Puri-
fication of the crude product using preparative TLC (eluent 10:1
CH2Cl2/MeOH) gave 62.1 mg (92.3%) of 27 as a white solid; mp
110–112 °C. 1H NMR (DMSO-d6, 400 MHz) d 0.98–1.36 (6H, m),
1.38–1.62 (2H, m), 1.72–2.06 (4H, m), 4.39 (2H, t, J = 6.8 Hz), 7.46
(1H, t, J = 6.3 Hz), 8.19 (1H, d, J = 8.0 Hz), 8.52 (1H, d, J = 4.6 Hz),
8.64 (1H, s), 8.70 (1H, s), 9.02 (1H, s), 10.31 (1H, s); 13C NMR
(DMSO-d6, 100 MHz) d 25.0, 25.7, 28.1, 28.4, 29.6, 32.2, 49.8,
121.9, 124.0, 126.8, 132.2, 143.5, 146.3, 148.8, 169.1. HRMS (FAB,
thioglycerol) calcd for [C15H21N5O2+H]+ 304.1773, found 304.1765.
Acknowledgments
We thank John Trott and Rajnish Sahu for their assistance with
the in vitro antimalarial and antileishmanial assays respectively.
This work was financially supported by Georgia Institute of Tech-
nology, by the Blanchard fellowship and by NIH Grant
R01CA131217 (A.K.O.). NCNPR is partially supported by USDA-
ARS scientific cooperative agreement no. 58-6408-2-0009. P.C.C.,
W.G. and B.G. are recipients of the GAANN predoctoral fellowship
from the Georgia Tech Center for Drug Design, Development and
Delivery.
4.2.25. 8-(p-N,N-Dimethylanilyl)triazolyloctahydroxamic acid
(23)
Deprotection of O-trityl-p-N,N-dimethylanilyltriazolyloctahydr-
oxamate 6 (186 mg, 0.32 mmol) under unoptimized conditions25
used BF3ꢀOEt2 (0.08 mL) without thioanisole in 4:1 CHCl3/MeOH
(15 mL) at rt for 3 h. The reaction was poured into 1:1 H2O/CHCl3
(100 mL) and the aqueous layer extracted with CHCl3 (50 mL).
The combined organic layer was washed with brine (50 mL), then
dried over Na2SO4 and concentrated in vacuo. Purification using
preparative TLC (eluent 12:1 CH2Cl2/MeOH) gave 43 mg (40%) of
23 as a white solid; mp 108–110 °C. 1H NMR (CD3OD, 400 MHz)
d 1.24–1.45 (6H, m), 1.49–1.68 (2H, m), 1.84–2.00 (2H, m), 2.00–
2.18 (2H, m), 2.97 (6H, s), 4.39 (2H, t, J = 7.1 Hz), 6.83 (2H, d,
J = 8.1 Hz), 7.65 (2H, d, J = 8.7 Hz), 8.12 (1H, s); 13C NMR (DMSO-
d6, 100 MHz) d 25.1, 25.8, 28.1, 28.4, 29.7, 32.2, 40.0, 49.4, 112.3,
118.8, 119.3, 126.0, 146.8, 149.9, 169.1. HRMS (FAB, thioglycerol)
calcd for [C18H27N5O2+H]+ 346.2243, found 346.2242.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.10.042.
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lL of
l
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